Method for detecting products derived from glucuronide metabolites with the enzyme b-glucuronidase, and a reagent comprising said enzyme

ABSTRACT

The present invention discloses a method and a highly efficient reagent to detect products derived from glucuronide metabolites presents in a sample, comprising the steps of adding to said sample an enzyme with β-glucuronidase activity originated from genus  Brachyspira  or any variant or mutant derived thereof; incubating the sample with the enzyme during a determined period of time; and detecting the product derived from glucuronide metabolite by means of a suitable technique. The reagent of the present invention comprises an enzyme with β-glucuronidase activity originated from genus  Brachyspira  or any variant or mutant derived thereof; and a suitable vehicle.

TECHNICAL FIELD

The present invention is related with the technical field ofbiotechnology, and particularly provides a method for detectingglucuronide derivatives present in a sample, using for this purpose aβ-glucuronidase enzyme from bacterial origin, as well as a reagentincluding said enzyme.

BACKGROUND OF THE INVENTION

Conjugation of endogenous and exogenous compounds with D-glucuronic acidis a metabolic pathway commonly known in animals and humans, which isnormally considered as a process for detoxification of organisms.Through this process, glucuronide conjugates or metabolites areproduced, which are excreted through urine or bile (Boppana V et al, J.Pharm. Sci. 1989. Vol 78(2):127-131). Compounds that are excreted inthese ways are mainly lipophilic, such as bilirubin, androgens,glucocorticoids, among others, and xenobiotic substances such asopioids, cannabinoids, benzodiazepines, among others.

Detection of these glucuronide metabolites in biological fluids allowsidentification and quantitation of endogenous or exogenous compoundspresent in the organism. Said detection can be performed by means ofchemical hydrolysis (acid hydrolysis) or by means of enzymatichydrolysis of said glucuronide metabolites. Chemical hydrolysis isperformed with an acid which hydrolyses most of conjugates in a relativereduced period of time (from 60 to 90 minutes) and at a low cost.However, the acid conditions and temperature required in this processdegradate sensitive compounds, for example, benzodiazepines. Moreover,working with acids in the laboratory requires stricter securityconditions, producing corrosion of vulnerable equipment. For thisreason, testing laboratories for these purposes mainly employ enzymatichydrolysis, consisting in the use of β-glucuronidases for hydrolyzationof glucuronide metabolites in conditions more manageable than acidhydrolysis.

Currently there exist diverse β-glucuronidase enzymes available fromdifferent origins, each one with optimum conditions for hydrolysis ofmetabolites. Thus, parameters such as concentration, pH, incubationperiod and temperature of reaction vary depending on the enzyme used.

For example, there exist alternatives of enzymes obtained from molluscssuch as a β-glucuronidase enzyme originated from Patella vulgatadescribed in document U.S. Pat. No. 4,473,640 of Combie et al, whichrequires a temperature between 60 and 70 degrees Celsius, pH 5, and atleast one hour to obtain only 60% of hydrolysis of the glucuronidemetabolite, and approximately three hours as the optimum time to reachcomplete quantification of the hydrolyzed metabolite. There also existβ-glucuronidase enzymes described from species Helix pomatia, Helixaspersa, Haliotis rufescens, among others (WO 95/16050, Scheinmann etal; Kemp et al, FAA. 2015. Report No. DOT/FAA/AM-15/6). Generally,preparations of enzymes obtained from molluscs tend to have morecontaminant substances that may interfere with further analysis ofglucuronide metabolites.

On the other hand, there also exist alternatives to enzymes frombacterial origin, such as β-glucuronidase from Escherichia coli.Comparative studies show that at an optimum pH and depending on the typeof glucuronide metabolite/substrate, it is required an hour ofincubation to obtain 40-80% of hydrolysis, and between three to fivehours to reach a complete hydrolysis (Wakabashi et al, J Biol. Chem.1961. Vol 236(4):996-1001). These detection times are excessive fortesting laboratories which analyze drugs in biological samples, and thesituation is even more dramatic in cases such as analysis of naturalopioids (codeine and morphine) and semi-synthetic opioids(hydromorphone, oxycodone and oxymorphone). The hydrolysis percentage ofconjugated metabolites of these opioids widely varies depending on theorigin of the enzyme used: after an incubation period of two hours ofthe biological sample with β-glucuronidase from Patella vulgata, only a21° A of codeine and a 64% of morphine are recovered; an 11° A ofcodeine and a 35% of morphine are obtained when β-glucuronidase fromHelix pomatia is used; and only a 9% of codeine and morphine arerecovered when β-glucuronidase from Escherichia coli is used (Wang etal., J Anal. Toxicol. 2006. Vol 30:570-575). For this reason, diverseresearchers seek enzymes capable of reduce the time needed to hydrolyzeglucuronide metabolites. The recently published patent application US2016/0090582 discloses a recombinant β-glucuronidase enzyme from E. coli(IMCSzyme™), which, according to the applicant, detects the presence ofdrugs in biological samples in 30 minutes or less, whose specificactivity is three times higher than wild type enzyme. However, in theexamples section it is shown that 60 minutes are required to incubatethe mutant enzyme with a sample containing codeine-6-glucuronide at 55degrees Celsius to recover 93.5% of codeine.

Therefore, new β-glucuronidase enzymes are needed to achieve quickhydrolysis of glucuronide metabolites and to obtain high recoverypercentages of their derivatives, in order to implement a highlyefficient method for detection and quantification of the same.

SUMMARY OF INVENTION

The present invention discloses a method to detect products derived fromglucuronide metabolites in a sample, comprising the steps of:

-   -   a) providing the sample in which said product is to be detected,    -   b) adding to said sample an enzyme with β-glucuronidase activity        from genus Brachyspira or any variant or mutant derived thereof,    -   c) incubating the sample with the enzyme for a determined period        of time; and    -   d) detecting the product derived from said glucuronide        metabolite through any suitable technique.

This method allows to detect any product derived from glucuronidemetabolites, whether from natural, semi-synthetic or synthetic origin.In a preferred embodiment, said derived products are selected from thegroup consisting of an opioid, an opiate, a cannabinoid, abenzodiazepine or any derivative thereof.

In a preferred embodiment, the sample from which these products derivedfrom glucuronide metabolites are detected, is selected from the groupconsisting of saliva, whole blood, plasma, urine, hair, skin, teeth,soft tissues, meconium, vitreous humor, water and food.

The enzyme with β-glucuronidase activity of the present invention, whoseamino acid sequence is shown in SEQ. ID. NO.1, preferably originatesfrom species Brachyspira pilosicoli. The invention also includes anyderived enzyme or mutant thereof, provided that it maintains itsβ-glucuronidase activity. Preferably, the mutant enzyme withβ-glucuronidase activity shares at least an 80% of identity with thesequence defined in SEQ. ID. NO.1.

In another preferred modality of the method of the present invention,incubation period of sample with the enzyme is of at least one minute,preferably between 2 and 120 minutes, and even more preferably between 3and 30 minutes. Incubation is performed in a range of temperaturebetween 20 and 60 degrees Celsius, preferably between 50 and 55 degreesCelsius, and in a pH range between 4.0 and 9.0, preferably between 6.5and 7.5.

Detection of products derived from glucuronide metabolites is performedthrough any suitable analytical technique, preferably selected from thegroup consisting of liquid chromatography (LC) or gas chromatography(GC), high performance liquid chromatography (HPLC), followed by massspectrometry (LC-MS, or GC-MS), or high resolution mass spectrometry(LC-HRMS, HPLC-HRMS), or tandem mass spectrometry (LC-MS-MS, GC-MS-MS,HPLC-MS-MS) or any other suitable technique or combination of detectionmethods.

A second object of the present invention is a reagent for detection ofproducts derived from glucuronide metabolites in a sample, whichcomprises an enzyme with β-glucuronidase activity originated from genusBrachyspira or any derived enzyme or mutant thereof; and an appropriatevehicle. Preferably, the reagent contains an enzyme with β-glucuronidaseactivity originated from species Brachyspira pilosicoli, whose aminoacid sequence is shown in SEQ. ID. NO.1, or any enzyme derived from saidsequence maintaining β-glucuronidase activity. Preferably, the derivedor mutant enzyme with β-glucuronidase activity shares at least an 80% ofidentity with the sequence defined in SEQ. ID. NO.1.

In a preferred embodiment, the reagent contains the enzyme in a suitablevehicle which is selected from the group consisting of water, salts,buffer solutions, Tris, HEPES, DTT, citric acid, EDTA, glycerol, sugars,amino acids or a combination thereof. Salts are preferably selected fromthe group consisting of sodium phosphate, potassium phosphate, sodiumcarbonate, sodium acetate, sodium citrate, sodium chloride and potassiumchloride. At the same time, sugars are selected from the groupconsisting of sorbitol, trehalose, sucrose, glucose, lactose, mannitoland raffinose.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows vector pET-28a with the optimized nucleotide sequenceencoding β-glucuronidase enzyme originated from B. pilosicoli to beexpressed in E. coli.

FIG. 2 shows an SDS-PAGE gel of β-glucuronidase enzyme purified withcolumn Ni-NTA.

FIG. 3 shows recovery of codeine and morphine versus time, usingβ-glucuronidase from B. pilosicoli.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for detection of productsderived from glucuronide metabolites present in a sample, comprising theuse of a new β-glucuronidase enzyme originated from Brachyspira genus.Hydrolysis efficiency of said enzyme is widely superior than allβ-glucuronidases currently known, with a specific activity 6.6 timeshigher than mean specific activity of a β-glucuronidase obtained from E.coli. This allows to detect products derived from glucuronidemetabolites in less than 15 minutes with a high recovery rate.

All the technical and scientific terms used to describe the presentinvention have the same meaning understood by any person with basicknowledge in the field under review. Nevertheless, to define moreclearly the scope of the invention, a list of terminology used in thisdescription is given below.

It should be understood that as used herein, the term “glucuronidemetabolite” refers to any compound conjugated with glucuronic acidthrough a glycosidic bond. It is to be understood that a glucuronidemetabolite can be natural or synthetic, and produced by chemical orenzymatic methods. The terms “conjugated compound”, “conjugatedmetabolite”, “glucuronide compound”, and “glucuronide substrate” areindistinctly used to describe the technical characteristics of thepresent invention.

The term “products derived from glucuronide metabolites” or “recoveredanalytes” should be understood as the molecule or original compound thatis produced after hydrolysis or glycosidic bond cleavage of aglucuronide metabolite. These derived products or original compounds canbe natural or synthetic, and produced by chemical or enzymatic methods.

The term “enzyme” should be understood as a sequence of amino acidswhich form a functional protein of interest for the present invention.It is to be understood that the present invention is not related withenzymes in their natural state, but it relates to proteins in anisolated state, purified or partially purified, or recombinant, obtainedby any method of genetic engineering known in the state of the art.Likewise, it is to be understood that the term “mutant” or “mutantenzyme” refers to a modified protein derived from the original aminoacid sequence maintaining its β-glucuronidase enzymatic activity; andsharing at least 70%, preferably 80% and more preferably 90% of identitywith the original sequence.

It is to be understood that the term “identity” among amino acidsequences refers to the percent of identical amino acids that thecompared sequences share among them, in a particular sequence alignmentwindow. The percentage of identity can be calculated using a sequencecomparison algorithm or by manual alignment together with visualinspection. For example, sequences and percentages of identity can beobtained using computer resources available on internet such as BLAST(http://blast.ncbi.nlm.nih.gov/) or FastDB computer programs.

The term “Unit (U)” should be understood as the unit used to measure thespecific activity of an enzyme. One unit will produce 1.0 μg ofphenolphthalein from phenolphthalein glucuronide (PPG) metabolite perhour at 37 degrees Celsius at pH 6.8.

The term “vehicle” should be understood as any substance that is addedto an active principle, in this case an enzyme, which is useful as asupport and allows to dilute or improve stability or durability of themain compound.

As described before, an object of the present invention relates to amethod for detection of glucuronide metabolites in a sample, whichcomprises the steps of adding an enzyme with β-glucuronidase activityfrom genus Brachyspira or any variant or mutant derived thereof to thesample in which a product derived from a glucuronide metabolite is to bedetected, incubating the sample with the enzyme for a determined periodof time; and detecting the product derived from said glucuronidemetabolite through any suitable technique.

This method is superior than others described in the state of the art,since it includes the use of the new enzyme with β-glucuronidaseactivity originated from genus Brachyspira, which has an improvedaffinity for glucuronide metabolites, a specific activity 6.6 timeshigher than β-glucuronidase originated from E. coli, and therefore it iscapable of hydrolyse glucuronide metabolites to be detected in a periodof time of at least one minute, preferably between three and 30 minutes.This enzyme is preferably originated from species Brachyspirapilosicoli, whose amino acid sequence is shown in SEQ. ID. NO.1, but itcan be obtained from any species of genus Brachyspira. Additionally, inthe method of the present invention, any derivative or mutant of thisprotein can be used, provided that it maintains its β-glucuronidaseactivity. Preferably, the derived or mutant enzyme with β-glucuronidaseactivity shares at least a 70% of sequence identity, more preferably an80% of identity, and even more preferably a 90% of identity with thesequence defined in SEQ. ID. NO.1.

The β-glucuronidase enzyme originated from Brachyspira can hydrolyse anyglucuronide metabolite, whether from natural, semi-synthetic orsynthetic origin. Some examples of these metabolites, withoutlimitation, are: morphine-3-glucuronide, morphine-6-glucuronide,codeine-6-glucuronide, oximorphone-glucuronide,hydromorphone-glucuronide, norbuprenorphine-glucuronide,buprenorphine-glucuronide, oxazepam-glucuronide, temazepam-glucuronide,lorazepam-glucuronide, alprazolam-glucuronide, midazolam-glucuronide,nordiazepam-glucuronide, psilocin-glucuronide,carboxy-tetrahydrocannabinol-glucuronide,tetrahydrocannabinol-glucuronide, tetrahydrocannabinolicacid-glucuronide, naloxone-3-glucuronide, tapentadol-glucuronide,tricyclic antidepressant-glucuronide, cannabidiol-glucuronide, amongothers. It is to be understood for the scope of the present inventionthat the β-glucuronidase enzyme originated from Brachyspira canhydrolise any glucuronide metabolite and it is not limited to theexamples previously mentioned.

Therefore, this method allows to detect any product derived fromglucuronide metabolites, whether from natural, semi-synthetic orsynthetic origin. For example, the method can be used to analyze drugs,toxic compounds derived from fungi, androgens, estrogens,mineralocorticoids, glucocorticoids, fatty acid derivatives, retinoids,among others.

In a preferred embodiment, products derived from glucuronide metabolitesare selected from the group consisting of opioids, opiates,cannabinoids, benzodiazepines or any other derived thereof. Preferably,the products derived from glucuronide metabolites are selected from thegroup consisting of morphine, codeine, oximorphone, hydromorphone,norbuprenorphine, buprenorphine, oxazepam, temazepam, lorazepam,alprazolam, midazolam, nordiazepam, psilocin,11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH), tetrahydrocannabinol(THC), tetrahydrocannabinolic acid (THCa), naloxone, tapentadol,tricyclic antidepressant, cannabidiol, among others, without beinglimited to the examples described herein.

In a preferred embodiment of the present invention, the method allows todetect any product derived from glucuronide metabolites in any type ofsample, which is preferably selected from the group consisting ofsaliva, whole blood, plasma, urine, hair, skin, teeth, soft tissues,water, food, meconium and vitreous humor, without restriction to thesetypes of samples. The following biological samples can also be used:bile, earwax, phlegm, vomit, aqueous humour, tears, amniotic fluid,vaginal secretion, semen, pre-ejaculate fluid, mucus, sebum, sweat,excrement, or any other suitable sample without limitation to the animalfluids previously mentioned. Other suitable samples originate from anon-animal source, such as food, water or chemical solutions fromlaboratories.

In the method of the present invention, β-glucuronidase enzymeoriginated from genus Brachyspira is added to the sample and the mixtureis incubated for a determined period of time, in suitable conditions. Ina preferred embodiment, incubation period of sample with the enzyme isof at least one minute, that is to say, only one minute of reaction isenough to detect the presence of a product derived of a glucuronidemetabolite in a sample. However, incubation period can be extended to 16hours or even more, provided that time is not an impediment to thetechnician performing the method.

More preferably, incubation period is between 2 and 120 minutes, andeven more preferably, incubation period is between 3 and 30 minutes,since obtaining the results efficiently and in the shortest time iscommonly desired. Additionally, adequate incubation conditions refer toideal conditions of temperature and pH in order to allow the reaction tobe produced in the time periods mentioned. Regarding temperature,incubation is performed preferably in a range between 20 and 60 degreesCelsius, and even more preferably in a range of 50 to 55 degreesCelsius. Regarding pH, incubation is performed in a range between 4.0and 9.0 and preferably between 6.5 and 7.5. As part of the elementsroutinely used in this assays, incubation is performed in presence ofbuffer solutions, standards of products derived from glucuronidemetabolites, polar or apolar solvents, among others.

After incubating the β-glucuronidase enzyme with the sample and beforeseparating the products derived from glucuronide metabolites throughseparation columns, an extraction step of said metabolites can beoptionally included to increase sensitivity of analytical techniques.This extraction step can be performed by means of liquid-liquidextraction (LLE), solid-liquid extraction (SLE), extraction in solidphase (SPE), or any other suitable technique. However, the step ofextracting the products derived from glucuronide metabolites adds alaborious step to the analytical method and increases operational costsdue to the use of solvents and other materials.

To the method of the present invention, the latter step is optional dueto the availability of highly selective analytical techniques that allowdirect analysis by means of the technique named “dilute and shoot”,which consist of diluting the sample with an adequate solution andinjecting it directly into a separation column. Moreover, the methoddoes not require elimination of the enzyme since the β-glucuronidase ofthe present invention is highly purified and has a high reactivity; andtherefore a small volume of the enzyme is required to react withglucuronide metabolites. In a preferred embodiment the volume range ofenzyme: liquid sample used, for example, urine, is from 1:1 to 1:14.Preferably, optimum concentration is between 0.02 and 0.25 mg of enzymeper mL of total reaction. In this manner, the volume of enzyme used doesnot affect or damage the separation column in case it is directlyinjected to it. The adequate solvent can be for example, water or amixture of water with methanol, or any mixture of solvents that can beused to dilute the sample.

Finally, detection of products derived from glucuronide metabolites isperformed through the use of separation columns and analyticaltechniques which are selected from the group consisting of: gaschromatography (GC), liquid chromatography (LC) or high performanceliquid chromatography (HPLC), solid phase microextraction (SPME), withmass spectrometry detectors (GC-MS; LC-MS; HPLC-MS; SPME-MS), highresolution mass spectrometry (GC-HRMS; LC-HRMS; HPLC-HRMS), tandem massspectrometry (GC-MS/MS; LC-MS/MS; SPME-GC-MS/MS), time of flight massspectrometry (LC-TOF-MS), ultraviolet-visible spectrophotometry(GC-UV/Vis; LC-UV/Vis; SPME-LC-UV/Vis), flame ionization detection(GC-FID; LC-FID; SPME-GC-FID), photodiode array detection, (LC-DAD), orany other suitable detection method, without limiting to the examplespreviously mentioned.

A second object of the present invention is a reagent for detection ofproducts derived from glucuronide metabolites in a sample, whichcomprises an enzyme with β-glucuronidase activity originated from genusBrachyspira or any derived enzyme or mutant thereof; which is includedin a suitable vehicle.

Preferably, the reagent contains an enzyme with β-glucuronidase activityoriginated from species Brachyspira pilosicoli, or any derived enzyme ormutant thereof. Said enzyme has an amino acid sequence shown in SEQ. ID.NO.1, but it can be any enzyme mutant from said sequence and maintainingβ-glucuronidase activity. Preferably, the derived or mutant enzyme withβ-glucuronidase activity shares at least a 70% or 80% of identity withthe sequence defined in SEQ. ID. NO.1, more preferably a 90% of identitywith the sequence defined in SEQ. ID. NO.1.

In a preferred embodiment, the reagent contains between 0.1 and 2 mg/mLof enzyme, in which the same has a β-glucuronidase activity from 200,000U/mL to 800.000 U/mL. In addition to β-glucuronidase enzyme, the reagentincludes an appropriate vehicle that varies depending on the productformat and on the function that the same performs. For example, thereagent including the enzyme can be in a liquid format, which couldrequire only water as a vehicle.

To improve the stability of the enzyme, salts can be added (atconcentrations μM or mM), or ethylenediamine tetraacetic acid (EDTA),Tris, dithiothreitol (DTT), hydroxyethylpiperazin-ylethanesulfonic acid(HEPES), citric acid, among others, or a combination thereof.Preferably, salts are selected from the group consisting of sodiumphosphate, potassium phosphate, sodium carbonate, sodium acetate, sodiumcitrate, sodium chloride and potassium chloride, without limitation tothe salts previously described. In case that the reagent is frozen, thevehicle to be used can include glycerol, preferably at a concentrationof 50%. On the other hand, if the reagent format is powder, which can beproduced by means of lyophilisation, spray drying or any otherpulverization method; vehicles able to protect the enzyme from dryingprocess are required such as sugars, amino acids and some salts, whichare present at a concentration range between 0.001 to 2% based on liquidprevious to the drying process. In this case, sugars are preferablyselected from the group consisting of sorbitol, trehalose, sucrose,glucose, lactose, mannitol and raffinose, without limitation to thesedescribed examples. Therefore, the vehicle of the present inventionfulfils the purpose of being a diluting, preservating or stabilizingmedium for the enzyme.

The following examples are considered to illustrate the invention andits preferred embodiments, but they must not be considered under anycircumstance as a restriction to the scope of the invention, which isdefined by the claims attached hereto.

EXAMPLES OF IMPLEMENTATION Example 1 Synthesis and Expression ofβ-glucuronidase Enzyme from B. pilosicoli

To express the β-glucuronidase enzyme, nucleotide sequence from saidprotein was used, originated from Brachyspira pilosicoli strain B2904described in Genbank (access code CP003490), which is shown in SEC. ID.NO.2, and codon optimization was performed using the algorithmOptimumGene™ (GenScript) to efficiently express said sequence in E.coli. To do this, codon adaptation index (CAI), understood asdistribution of frequency of codon usage through the sequence, wasadjusted from 0.66 to 0.88, in which a CAI value of 1.0 is considered tobe perfect in the desired expression organism; and a CAI value greaterthan 0.8 is regarded as good in terms of high gene expression levels.Guanine-cytosine content was also optimized to extend mRNA half-life andstem-loop structures were eliminated, since they disrupt ribosomebinding and mRNA stability. Optimized nucleotide sequence obtained isshown in SEC. ID. NO.3. Amino acid sequence of the enzyme is shown inSEC. ID. NO.1.

The optimized nucleotide sequence of the enzyme was cloned into vectorpET-28a, thus vector pET-28a-glucuronidase was obtained, which is shownin FIG. 1. To express β-glucuronidase enzyme from B. pilosicoli in E.coli, the following protocol was carried out: 5 μL of competent E. colistrain BL21 cells were incubated in ice with 10 ng ofpET-28a-glucuronidase vector during 20 minutes. Then, bacterial cellswere submitted to heatshock at 37 degrees Celsius during 3 minutes.Bacterial cells were then put in ice for 30 seconds and later cells wererecovered with 250 μL of LB io medium at 37 degrees Celsius and platedin LB-agar with 50 mg/L of kanamycin, and colonies were grown. Finally,a β-glucuronidase activity test was carried out on plates with IPTG(isopropyl-β-thiogalactoside) and X-Gal (5-bromo-4-chloro-3indolyl-beta-D-thiogalactopyranoside).

In order to purify the enzyme, E. coli BL21 transformed cells were grownin 500 mL of LB medium supplemented with kanamycin (50 mg/L), untilobtention of an optical density of 0.3, measured using aspectrophotometer at 600 nm (DO₆₀₀).

Then, IPTG at a concentration of 1 mM was added to the culture and thenincubated at 37 degrees Celsius during 6 hours. Cells were subjected tosonication during 3 minutes with an amplitude of 60 (cycles of 15seconds in ice). Later, the enzyme was purified by means of a system forprotein purification in columns with Ni-NTA (Nickel-Nitriloacetic acid)resins with elution of 2 mL of Tris-HCl 50 mM, pH 5.6 with imidazole 100mM pH 5.9 or with imidazole 500 mM pH 4.5.

FIG. 2 shows an SDS-PAGE gel of purified proteins in column Ni-NTA. Eachwell contained proteins at a concentration of 15 pg/mL, except in crudeextract (E. coli pET28a-glucuronidase without purification in thecolumn), in which 5 μL were loaded. The acronym Std corresponds tomolecular weight standard in kilodaltons (kD), Crude ex corresponds tocrude extract, Glu E1 corresponds to elution with imidazole 100 mM pH5.9 and Glu E2 corresponds to elution with imidazole 500 mM pH 4.5.

Example 2 Incubation Time of a Sample with β-glucuronidase from B.pilosicoli

The required time to hydrolyze morphine-3-glucuronide andcodeine-6-glucuronide with the enzyme β-glucuronidase in excess (namedBGTurbo™) from B. pilosicoli was evaluated with the following protocol:10 μL of glucuronide metabolites at a concentration of 100 μg/mL, freestandards (free codeine and morphine calibrators at concentrations of312.5, 625, 1,000, 1,250 and 1,875 ng/mL), and internal standards (40 μLof deuterated free drug at a concentration of 1 μg/mL to each sample)were added to a sample of 0.4 mL of blank urine and then submitted tovortex agitation. Then, 360 μL of sodium phosphate (pH 7; 140 mM)solution was added to each sample. Then, 240 μL of β-glucuronidase(BGTurbo™) were added to each sample and then agitated by vortex.Samples were incubated at 55 degrees Celsius in an oven during 0, 10,20, 30, 40, 50 and 60 minutes. A total of 21 samples were used: 7 pointsof measure at different times, each one in triplicate. Calibrators wereincubated during 30 minutes. The samples incubated at different timeswere removed from oven and the reaction was stopped by adding 700 μL ofsodium phosphate buffer solution 1, 5 M and agitated by vortex. Then, pH8-9 was verified with pH paper, and 2 mL of chloroform: isopropanol50:50 were added to samples, and they were agitated by vortex during 2minutes. Aqueous phase was discarded and organic phase was transferredto 12×75 mm assay tubes and then evaporated under nitrogen gas (in awater bath at 50 degrees Celsius) until approximately 1 mL. Samples werethen agitated by vortex previous to evaporation until dryness. Sampleswere reconstituted in 1 mL of HCl 0, 5 N in methanol for LC-MS/MSanalysis.

Calculation of concentration of equivalents was performed as follows:

Morphine:

-   -   Molecular weight of morphine: 285.34 g/mol    -   Molecular weight of morphine-3-glucuronide: 461.462 g/mol    -   Proportion of molecular weight of        morphine/morphine-3-glucuronide:

Morphine equivalents:

${{{0.61832},500\mspace{14mu} \frac{ng}{mL}\mspace{14mu} {Morphine}} - 3 - {glucuronide}} = {1,545.8\mspace{14mu} \frac{ng}{mL}\mspace{14mu} {Morphine}}$

Codeine:

-   -   Molecular weight of codeine: 299.364 g/mol    -   Molecular weight of codeine-6-glucuronide: 475.49 g/mol    -   Proportion of molecular weight of codeine/codeine-6-glucuronide:

Codeine equivalents:

${{{0.629592},500\mspace{14mu} \frac{ng}{mL}\mspace{14mu} {Codeine}} - 6 - {glucuronide}} = {1,574.0\mspace{14mu} \frac{ng}{mL}\mspace{14mu} {Codeine}}$

Table 1 and FIG. 3 show the results obtained. Hydrolysis of 100% ofcodeine-6-glucuronide was obtained in less than 10 minutes. In the caseof morphin, approximately 65% of hydrolysis was obtained within 10minutes, but these values can be improved by optimizing parameters oftemperature, pH or the method to stop reaction. Due to the use ofinternal controls for recovery of products derived from glucuronidemetabolites, values have a standard deviation up to 20%.

TABLE 1 Tabulated results of FIG. 3. Time Codeine % Yield Morphine %Yield (minutes) (ng/mL) Codeine (ng/mL) Morphine 0 30.0 1.9 195.8 12.710 1826.9 116.1 997.4 64.5 20 1867.5 118.6 969.2 62.7 30 1694.3 107.6871.2 56.4 40 1821.0 115.7 811.5 52.5 50 1844.3 117.2 847.4 54.8 601819.5 115.6 823.4 53.3

Example 3 Calculation of Specific Activity of β-glucuronidase from B.pilosicoli

Specific activity of the β-glucuronidase enzyme from B. pilosicoli(BGTurbo™) was calculated and compared with a mean value of threecalculations of the specific activity of β-glucuronidase from E. coli,using the following protocol: 350 μL of sodium phosphate buffer solution(NaH₂PO₄) 0.1 M, pH 6.8 were mixed with 350 μL of phenolphthaleinβ-D-glucuronide sodium salt (0.64 mg/mL) and the mixture was incubatedat 37 degrees Celsius. Then, 50 μL of solution containing the enzymewere added to the mixture and incubated at 37 degrees Celsius during 30minutes. After io the first 15 minutes the mixture was gently agitatedand stopped at 30 minutes from the beginning of the reaction with 2.5 mLof a glycine solution 0.2 M, pH 10.4. Absorbance was read at 540 nm andthe amount of enzyme used was calculated (U/mL) when adjusting thevalues in a calibration curve. Protein concentration was measured usingBradford method, as widely known in literature. As shown in Table 2,results indicate that BGTurbo™ enzyme has a specific activity 6.6 timeshigher than mean specific activity of β-glucuronidase from E. coli.

TABLE 2 Enzymatic activity of β-glucuronidase from B. pilosicoli.Activity Proteins Specific Activity Enzyme (U/mL) (mg/mL) (U/mg)BGTurbo ™ 427,805 2.08 205,676 E. coli 1 68,779 2.28 30,166 E. coli 2102,710 3.17 32,401 E. coli 3 85,084 2.89 29,441 E. coli mean value30,669

Example 4 Comparison of β-glucuronidase from B. pilosicoli withβ-glucuronidases from Different Sources

To test the efficiency of β-glucuronidase from B. pilosicoli (BGTurbo™),this enzyme was compared with two β-glucuronidases currently availablein the market: DR2102 (Campbell Science) and BG100™ (KURA Biotec). To dothis, 300 ng of each of the glucuronide metabolites of buprenorphine,codeine, hydromorphone, lorazepam, morphine, oxymorphone and11-nor-9-carboxy-tetrahydrocannnabinol (THC-COON) were added to a sampleof blank urine, therefore obtaining spiked urine. Each enzyme was usedunder the conditions proposed by each manufacturer. Hydrolysis of 50 μLof urine was performed, diluting it with 250 μL of buffer solution at pH6.8, 20 μL of internal standards (ISTD) and 10 μL of DR2102 or BG100enzymes, or 20 μL of BGTurbo™. Incubation conditions are shown in Table3.

TABLE 3 Conditions used for each enzyme. Conditions DR2102 BG100 ™BGTurbo ™ Urine (μL) 50 50 50 ISTD (μL) 20 20 20 Buffer (μL) 25 25 25Enzyme (μL) 10 10 20 Temperature (° C.) 62 68 50 Time (minutes) 60 30 15

After hydrolysis, samples were centrifuged at 24,000 g, diluted with 450μL of a solution comprising 95% water and 5% methanol, and charged to anHPLC column (Phenomenex). Protein amount in samples was measured beforeand after centrifugation at 24,000 g using a NanoDrop spectrophotometer(Thermo Scientific). As a result, the reactions performed were carriedout with 1.787 mg/mL of DR2102, 0.554 mg/mL of BG100™ and 0.103 mg/mL ofBGTurbo™.

Recovery yields of products derived from glucuronide metabolites areshown in Table 4. As shown, mean recovery of derived products wasimproved in a 15% when hydrolysis was performed with BGTurbo™ in only 15minutes and using 70% and 94% less of protein amount in comparison tothe other two enzymes. It is interesting to mention that β-glucuronidaseenzyme from B. pilosicoli is more reactive with codeine-6-glucuronidethan the other enzymes.

TABLE 4 Results of analites recovered from hydrolysis of glucuronidemetabolites with three β-glucuronidases enzymes from different originsDR2102 BG100 ™ BGTurbo ™ 60 minutes 30 minutes 15 minutes RecoveredAnalyte (ng/mL) (ng/mL) (ng/mL) Buprenorphine 223.1 271.1 224.4 Codeine159.4 173.8 243.2 Hydromorphone 176.8 196.0 196.2 Lorazepam 214.9 202.5185.9 Morphine 174.5 190.9 198.1 Oximorphone 155.8 189.0 169.4 THC-COOH75.5 146.0 140.6 Protein in sample 1.787 (mg/mL) 0.554 (mg/mL) 0.103(mg/mL) pre-centrifugation Protein in sample 0.725 (mg/mL) 0.280 (mg/mL)0.085 (mg/mL) post-centrifugation and injected on HPLC column

Example 5 Comparison of β-glucuronidase from B. pilosicoli with Otherβ-glucuronidases from Bacterial Origin

In order to test enzyme speed of β-glucuronidase from B. pilosicoli(BGTurbo™) in comparison with β-glucuronidases from bacterial origin,BGTurbo™ was compared with EBG enzyme (recombinant β-glucuronidase fromE. coli, KURA Biotec) and with IMCSzyme™ (IntegratedMicro-Chromatography Systems) at different incubation periods andconcentration. The method previously described was used, but analyzingonly hydrolysis of codeine-6-glucuronide and morphine-3-glucuronide withthe conditions described in Table 5.

TABLE 5 Conditions used for each enzyme. BGTurbo ™ EBG IMCSzyme ™Control Conditions A B C D F — E Buffer (μL) 360 360 360 360 360 360 360Water (μL) 0 120 180 210 180 180 120 Spiked urine (μL) 400 400 400 400400 400 400 Enzyme (μL) 240 120 60 30 60 60 0 Internal Standard (μL) 4040 40 40 40 40 40 Storage buffer control (μL) 0 0 0 0 0 0 120 TotalVolume (μL) 1,040 1,040 1,040 1,040 1,040 1,040 1,040 Incubation Time(minutes) 10 10 10 10 0, 10, 20 0, 10, 20 10 pH 7.0 7.0 7.0 7.0 7.0 7.07.0 Temperature (° C.) 55 55 55 55 55 55 55

Each condition shown in Table 5 was assayed in duplicate, and eachanalysis was measured in triplicate. Calculation of concentration ofequivalents was performed as described in Example 2.

Table 6 shows the results obtained from analysis of codeine, and Table 7shows results obtained from analysis of morphine. As can be noticed,codeine-6-glucuronide was hydrolyzed in a mean value of 109,59% within10 minutes, using 240 μL of BGTurbo™. The value mentioned is greaterthan 100% because standards of glucuronide metabolites used in theanalysis are not completely pure, and present a fraction of the originalcompound that is added to the final result. On the other hand, whencomparing hydrolysis yield of codeine-6-glucuronide andmorphine-3-glucuronide using the same volume of the enzymes (60 μL), itis shown that BGTurbo™ is better than the other EBG and IMCSzyme™enzymes within 10 minutes. Even after an incubation period of 20 minutesof the samples with EBG and IMCSzyme™, these enzymes could not producethe same hydrolysis yield that was obtained with BGTurbo™ within 10minutes.

TABLE 6 Results of codeine analysis. Condition/ Codeine Codeine Averageof Assay Sample (ng/mL) % Yield Assay Yield (%) A/1 BGTurbo ™ (10 min,240 μL) 1,740 110.55 109.59 A/2 BGTurbo ™ (10 min, 240 μL) 1,710 108.64B/1 BGTurbo ™ (10 min, 120 μL) 1,270 80.69 78.14 B/2 BGTurbo ™ (10 min,120 μL) 1,190 75.60 C/1 BGTurbo ™ (10 min, 60 μL) 879 55.84 54.19 C/2BGTurbo ™ (10 min, 60 μL) 827 52.54 D/1 BGTurbo ™ (10 min, 30 μL) 23014.61 22.20 D/2 BGTurbo ™ (10 min, 30 μL) 469 29.80 F/1 EBG (0 min, 60μL) No Peak 0.00 0.00 F/2 EBG (0 min, 60 μL) No Peak 0.00 F/1 EBG (10min, 60 μL) 154 9.78 10.48 F/2 EBG (10 min, 60 μL) 176 11.18 F/1 EBG (20min, 60 μL) 396 25.16 27.10 F/2 EBG (20 min, 60 μL) 457 29.03 1IMCSzyme ™ (0 min, 60 μL) No Peak 0.00 0.00 2 IMCSzyme ™ (0 min, 60 μL)No Peak 0.00 1 IMCSzyme ™ (10 min, 60 μL) 189 12.01 11.85 2 IMCSzyme ™(10 min, 60 μL) 184 11.69 1 IMCSzyme ™ (20 min, 60 μL) 395 25.10 27.26 2IMCSzyme ™ (20 min, 60 μL) 463 29.42 E/1 Control (10 min) No Peak 0.000.00 E/2 Control (10 min) No Peak 0.00

TABLE 7 Results of the morphine analysis. Condition/ Morphine MorphineAverage of Assay Sample (ng/mL) % Yield Assay Yield (%) A/1 BGTurbo ™(10 min, 240 μL) 796 51.49 51.49 A/2 BGTurbo ™ (10 min, 240 μL) 79651.49 B/1 BGTurbo ™ (10 min, 120 μL) 717 46.38 46.90 B/2 BGTurbo ™ (10min, 120 μL) 733 47.42 C/1 BGTurbo ™ (10 min, 60 μL) 687 44.44 43.70 C/2BGTurbo ™ (10 min, 60 μL) 664 42.96 D/1 BGTurbo ™ (10 min, 30 μL) 49832.22 38.46 D/2 BGTurbo ™ (10 min, 30 μL) 691 44.70 F/1 EBG (0 min, 60μL) 179 11.58 12.71 F/2 EBG (0 min, 60 μL) 214 13.84 F/1 EBG (10 min, 60μL) 609 39.40 39.62 F/2 EBG (10 min, 60 μL) 616 39.85 F/1 EBG (20 min,60 μL) 629 40.69 43.47 F/2 EBG (20 min, 60 μL) 715 46.25 1 IMCSzyme ™ (0min, 60 μL) 142 9.19 10.19 2 IMCSzyme ™ (0 min, 60 μL) 173 11.19 1IMCSzyme ™ (10 min, 60 μL) 603 39.01 39.20 2 IMCSzyme ™ (10 min, 60 μL)609 39.40 1 IMCSzyme ™ (20 min, 60 μL) 662 42.83 43.41 2 IMCSzyme ™ (20min, 60 μL) 680 43.99 E/1 Control (10 min) No Peak 0.00 0.00 E/2 Control(10 min) No Peak 0.00

1. A method to detect products derived from glucuronide metabolites in asample, comprising the steps of: a) providing the sample in which saidproduct is to be detected, b) adding to said sample an enzyme withβ-glucuronidase activity from genus Brachyspira or any variant or mutantderived thereof, c) incubating the sample with the enzyme for adetermined period of time; and d) detecting the product derived fromsaid glucuronide metabolite through any suitable technique.
 2. Themethod of claim 1, wherein products derived from glucuronide metabolitesare from natural, semi-synthetic or synthetic origin.
 3. The method ofclaim 2, wherein products derived from glucuronide metabolites areselected from the group consisting of an opioid, an opiate, acannabinoid, a benzodiazepine or any derivative thereof.
 4. The methodof claim 1, wherein the sample is a biological sample.
 5. The method ofclaim 4, wherein the biological sample is selected from the groupconsisting of saliva, whole blood, plasma, urine, hair, skin, teeth,soft tissues, meconium and vitreous humor.
 6. The method of claim 1,wherein the enzyme with β-glucuronidase activity originates from speciesBrachyspira pilosicoh.
 7. The method of claim 6, wherein the enzyme withβ-glucuronidase activity is selected from the group consisting of: a) anamino acid sequence comprising the amino acid sequence shown in SEQ. ID.NO.1; b) an amino acid sequence sharing at least a 70% of identity withthe amino acid sequence defined in a).
 8. The method of claim 1, whereinthe incubation period of the sample and the enzyme is between 1 and 30minutes.
 9. The method of claim 1, wherein incubation of sample andenzyme is performed at a temperature range between 20 and 60 degreesCelsius.
 10. The method of claim 1, wherein incubation of sample andenzyme is performed at a range of pH between 4.0 and 9.0.
 11. The methodof claim 1, wherein the suitable technique to detect products derivedfrom glucuronide metabolites is selected from the group consisting ofLC-MS, GC-MS, HPLC-MS, LC-HRMS, HPLC-HRMS, LC-MS-MS, GC-MS-MS andHPLC-MS-MS.
 12. A reagent to detect products derived from glucuronidemetabolites in a sample, comprising the steps of: a) an enzyme withβ-glucuronidase activity originated from genus Brachyspira or anyvariant or mutant derived thereof; and b) a suitable vehicle.
 13. Thereagent of claim 12, wherein the enzyme with β-glucuronidase activityoriginates from species Brachyspira pilosicoli.
 14. The reagent of claim13, wherein the enzyme with β-glucuronidase activity is selected fromthe group consisting of: a) an amino acid sequence comprising the aminoacid sequence shown in SEQ. ID. NO.1; b) an amino acid sequence sharingat least a 70% of identity with the amino acid sequence defined in a).15. The reagent of claim 12, wherein the suitable vehicle is selectedfrom the group consisting of: water, salts, buffer solutions, Tris,HEPES, DTT, citric acid, EDTA, glycerol, sugars, amino acids or acombination thereof.
 16. The reagent of claim 15, wherein the salts areselected from the group consisting of: sodium phosphate, potassiumphosphate, sodium carbonate, sodium acetate, sodium citrate, sodiumchloride and potassium chloride.
 17. The reagent of claim 15, whereinsugars are selected from the group consisting of sorbitol, trehalose,sucrose, glucose, lactose, mannitol and raffinose.